The Notch signaling pathway plays multiple critical roles in embryonic development, including cell type specification, differentiation, and patterning. In the thymus, Notch signaling regulates key stages of T lineage determination and thymocyte differentiation. Notch signaling has also been implicated in regulating fetal TEC differentiation i a fetal thymic organ culture (FTOC)-based assay, although Notch signaling in TECs was not directly proven. In hair follicle development, Notch1 is a direct downstream target of Foxn1, which is the gene mutated in nude mice and is also a key transcription factor regulating multiple stages in TEC differentiation in both the fetal and postnatal thymus. We recently discovered that Notch1 is specifically expressed in a subset of fetal TECs that co-label with Foxn1 and Plet-1, a TEC progenitor marker. Deletion of Notch1 in TECs using a Foxn1Cre allele results in depletion of the TEC progenitor markers Plet-1, Cld3/4, and UEA-1 during fetal stages and the presence of large epithelial-free zones in the 1-month postnatal thymus. T-lineage specification is unaltered, and thymocyte numbers and differentiation are surprisingly normal. These phenotypes are strikingly similar to the published phenotype for deletion of the Kremen1 Wnt inhibitor, which results in a mild up-regulation of Wnt signaling. Wnt signaling has also been implicated in TEC differentiation, although the evidence has been conflicting. As Notch signaling has been demonstrated in other systems to be capable of directly inhibiting the canonical Wnt signaling pathway, the similarity between TEC-specific Notch deletion and Wnt up-regulation suggests that Notch1 may be acting in part to modulate Wnt signaling in TECs. In this proposal, we will build on these preliminary observations to investigate the role of Notch signaling in TECs in the fetal and postnatal thymus. We will determine the ontogeny of the epithelial-free zones in the TEC-specific Notch1 mutant thymus, and determine how this phenotype progresses with aging. We will determine whether this altered stromal structure affects specific aspects of thymocyte differentiation, including later stages of thymocyte differentiation and export. Finally, we will test whether some or all of these phenotypes are due to Notch1 modulation of the canonical Wnt signaling pathway in TECs. These experiments will provide essential and critical information that will test our preliminary hypothesis that Notch1 acts in TEC progenitors to regulate the balance between self-renewal and differentiation, in part by modulating the response to Wnt signaling.